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. 2013 Apr 14;112(4):741–749. doi: 10.1093/aob/mct070

Environmental influences on growth and defence responses of the invasive shrub, Lonicera maackii, to simulated and real herbivory in the juvenile stage

Deah Lieurance 1,*, Don Cipollini 1
PMCID: PMC3736768  PMID: 23589632

Abstract

Background and Aims

Tolerance and defence against herbivory are among the many mechanisms attributed to the success of invasive plants in their novel ranges. Because tolerance and defence against herbivory differ with the ontogeny of a plant, the effects of herbivore damage on plant fitness vary with ontogenetic stage and are compounded throughout a plant's lifetime. Environmental stresses such as light and nutrient limitations can further influence the response of the plant. Much is known about the response of plants in the seedling and reproductive adult stages, but less attention has been given to the pre-reproductive juvenile stage.

Methods

Juvenile plants of the North American invasive Lonicera maackii were exposed to simulated herbivory under high and low light and nitrogen availability and growth, allocation patterns and foliar defensive chemistry were measured. In a second experiment, complete nutrient availability and damage type (generalist caterpillar or simulated) were manipulated.

Key Results

Juvenile plants receiving 50 % defoliation had lower total biomass and a higher root^:^shoot ratio than controls for all treatment combinations except low nitrogen/low light. Low light and defoliation increased root^:^shoot ratio. Light, fertilization and defoliation had little impact on foliar defensive chemistry. In the second experiment, there was a reduction in total biomass when caterpillar damage was applied. The root^:^shoot ratio increased under low soil fertility and was not affected by defoliation. Stem-diameter growth rates and specific leaf area did not vary by damage type or fertilization. Foliar protein increased through time, and more strongly in defoliated plants than in controls, while peroxidase activity and total flavonoids decreased with time. Overall, resource limitations were more influential than damage in the growth of juvenile L. maackii plants.

Conclusions

The findings illustrate that even when resources are limited, the tolerance and defence against herbivory of a woody invasive plant in the juvenile stage may contribute to the establishment and persistence of some species in a variety of habitats.

Keywords: Herbivory, Lonicera maackii, tolerance, root to shoot ratio, relative growth rate, ontogeny

INTRODUCTION

When considering plant–herbivore interactions in the context of exotic plant invasions, most research focuses on the release from herbivore pressure as predicted by the enemy release hypothesis (Liu et al., 2007; Adams et al., 2009; Funk and Throop, 2010; Andonian and Hierro, 2012). But how an invasive plant responds to natural amounts of herbivory experienced in the novel habitat and potential major defoliation events is not as thoroughly explored. The ability to tolerate defoliation may contribute to invasive success since tolerance could be beneficial to invasive plant establishment if they remain susceptible to herbivore attack in the new range. Although defoliation tolerance is relatively poorly understood for non-native plant species, there is some evidence that the ability to re-grow following herbivore damage is important (Barton and Koricheva, 2010; Hanley, 2012 and references therein). Tolerance is exhibited through several mechanisms contributing to plant fitness following damage, including increases in physiological performance, reallocation of resources to compensate for tissue removal, and modified root^:^shoot ratios that lead to improved nutrient uptake (Karban and Baldwin, 1997; Strauss and Agrawal, 1999; Haukioja and Koricheva, 2000). It has been suggested that a trade-off between growth and resistance may determine how a plant responds to herbivory and these responses can vary with resource availability (van der Meijden et al., 1988; Fineblum and Rausher, 1995). However, it is possible that plants could respond to herbivore damage with a mixed response in both tolerance and resistance to herbivory (Koricheva et al., 2004; Núñez-Farfán et al., 2007; Agrawal, 2010). Perhaps a trait of highly successful invasive plants is being a ‘jack of all trades’ in the face of herbivore pressure (Koricheva et al., 2004).

Ontogeny may also play a part in the response of a plant to damage, as allocation to growth, defence and reproduction is variable with age (Fritz et al., 2001; Boege, 2005; Boege and Marquis, 2006; Hanley et al., 2007). Pre-reproductive seedling and juvenile plants are often more vulnerable to herbivory and damage incurred early in development can influence establishment, growth and, ultimately, the reproductive success of the plant later in life (Hanley and May, 2006; Boege et al., 2007; Hanley et al., 2007). Furthermore, these responses differ between annual, herbaceous plants and perennial, woody plants where long-lived plants can cope with damage by offsetting reproduction and re-deploying limiting resources to ensure longevity, and annual plants employ strategies to maximize reproductive fitness (Strauss and Agrawal, 1999; Barton and Koricheva, 2010).

Differences in abiotic factors such as light and nutrient availability can alter the tolerance and/or resistance of a plant to herbivore damage. Reducing light availability to plants limits carbon directly through reduction in photosynthesis and, for some woody plants, may influence its ability to recover from herbivory. For example, when the tolerance of ten Dipterocarpaceae species was evaluated, removal of 35 % of the leaf area from these species reduced relative growth rate (RGR) by approx. 22 % for seedlings grown in deep shade, but only by approx. 9 % for those in high light, when compared with undamaged plants (Paine et al., 2012). Additionally, woody plants growing in high-nutrient conditions may favour growth and place higher allocation to above-ground components at the expense of below-ground carbon stores that can be tapped when plants respond to herbivore damage (Hawkes and Sullivan, 2001). This may result in reduced tolerance in high-nutrient treatments. Alternatively, increases in nutrient availability may provide plants with adequate resources to rapidly compensate for leaf loss to herbivory (Maschinski and Whitham, 1989). Secondary metabolite production also varies with resource availability (Bryant et al., 1983; Koricheva et al., 1998; Herms, 2002). Phenolics typically increase with light availability and not only offer defence against herbivores, but also protect against photodamage (Dudt and Shure, 1994; Close and McArthur, 2002). Additionally, the activity of peroxidase, an inducible enzyme linked to defence, has been shown to decline with nitrogen limitation while protein content increases with nitrogen enrichment (Dietrich et al., 2004).

Lonicera maackii is a deciduous, woody shrub native to south-east Asia that is highly invasive throughout the Midwestern USA (Luken and Thieret, 1996). Traits thought to contribute to the success of L. maackii include extended leaf longevity, high above-ground growth rates and fecundity, broad phenotypic plasticity, and tolerance to a variety of habitats (Luken et al., 1995a, b, 1997a, b; Trisel, 1997; Lieurance, 2004). Current land-use patterns provide disturbed and fragmented forest habitats in addition to newly established early successional old field sites wherein L. maackii may comprise up to 50 % of the understorey species pool (Medley, 1997; Hartman and McCarthy, 2008). Plants typically lose only 3 % or less of their leaf area per year to arthropod herbivores (Lieurance and Cipollini, 2012, 2013), but it can occasionally receive much higher amounts of leaf area loss through browsing by deer or other herbivores. However, no studies of tolerance to foliar herbivory have been conducted on L. maackii to determine what threshold of natural herbivory is biologically significant.

Several secondary metabolites associated with resistance to herbivores have been identified in L. maackii, including apigenin and luteolin, their glycoside derivatives, chlorogenic acid and other phenolics in the leaves (Cipollini et al., 2008b). Such compounds have been implicated in allelopathic effects on other plants, as well as serving an anti-herbivore function (Dorning and Cipollini, 2006; Cipollini et al., 2008a, b). Secondary metabolites are known to be constitutively produced, but it is not known how components of the defensive chemistry of L. maackii may change after herbivory, respond to environmental variation or correlate with growth.

We examined growth and biochemical responses of juvenile L. maackii plants to herbivory, and how abiotic factors (light and nutrient availability) influenced these responses in two greenhouse experiments. We predicted that (a) juvenile L. maackii plants would be tolerant of both real and artificial herbivore damage; (b) tolerance would be higher in high-resource conditions than in low-resource conditions; and (c) measures of foliar defensive chemistry would increase with herbivore damage, protein and peroxidase would decrease with nutrient limitation, flavonoids would decrease with light limitation, and all would increase through time.

MATERIALS AND METHODS

Effects of simulated herbivory in high- and low-light and nitrogen treatments

Lonicera maackii (Rupr.) Maxim seeds were collected in 2008 from Wright State University Woods (39·78766 N, 84·05665 W) and stratified for 5 weeks at 22 °C. Germinants were transplanted from Petri dishes to 400-mL plastic round pots in ProMix BX potting soil with mychorrhizae added (Premier Tech Horticulture, Quakertown, PA, USA). Plants were watered with distilled water as needed and fertilized once every 2 weeks with 125 mL of 1·87 g L−1 Peters 20–20–20 complete soluble fertilizer plus micronutrients (Grace-Sierra, Milpitas, CA, USA) until the start of the experiment.

Once the plants reached 12 weeks of age, treatments including simulated herbivory (0 % and 50 % removed), light availability (100 % and 50 % ambient light), and nitrogen availability (full- and half-strength nitrogen fertilization) were assigned to plants in a three-way factorial design with eight replicates per combination for a total of 64 plants. Juvenile plants were on average 25 cm high and had approx. 20 leaves per plant at the start of the experiment. The simulated herbivory treatment was imposed through a one-time manual removal with scissors at the petiole of 50 % of the leaves on the plant, alternating every other leaf (plants generally had six to ten leaves removed). While low levels of arthropod herbivory are commonly observed on L. maackii in the field (Lieurance and Cipollini, 2012, 2013), plants occasionally receive large amounts of herbivory by deer or other herbivores. Mature shrubs tolerate loss of up to 50 % of their leaf area in a single bout with little effect on branch growth rates (Lieurance, 2012), but it is unknown how higher amounts of herbivory may affect juvenile plants. Exposure of plants to high levels of damage should provide a conservative estimate of their tolerance capacity. Plants in the shaded treatment were grown in shade structures (three structures, 10 or 11 plants per structure) constructed with PVC pipe and black polypropylene shade cloth (DeWitt Co., Sikeston, MO, USA) that reduced light by 50 %, while plants in the unshaded treatment received ambient light supplemented with fluorescent lights. Ambient light conditions ranged from 0700 to 2100 µmol m−2 s−1 of photosynthetically active radiation. The nitrogen treatment was implemented by fertilizing plants bi-weekly with 200 mL of complete nutrient solution with 1500 ppm nitrogen for the high-nitrogen treatment and a complete nutrient solution with 750 ppm nitrogen concentration for the low-nitrogen treatment. Nutrient solutions were prepared as in Reiss (1994). To avoid possible microclimatic effects in the greenhouse, plants were rotated within the shaded and unshaded treatments every 2 weeks. All plants were watered with distilled water as needed between fertilization treatments. Stem diameter at base (DAB) and height were measured every 2 weeks for the duration of the experiment from the initiation of the treatments.

Fourteen weeks after initiation of the treatments, we harvested plants and divided them into roots and shoots. Roots were washed with distilled water to remove soil and biomass was dried to a constant weight at 70 °C for a minimum of 72 h and weighed. Total dry biomass, and the mass of the component parts were recorded. The root : shoot ratio (R:S) was calculated as dry mass of roots/dry mass of above-ground biomass (stems and leaves). Relative growth rate (RGR) in basal stem diameter was calculated as RGR = [ln(DABfinal) – ln(DABini)]/number of days. Mean specific leaf area (SLA) was calculated from three leaves per plant as leaf area (cm2)/g dry leaf mass. Prior to harvesting, whole fresh leaf (approx. 500 mg) samples were collected randomly for chemical analysis and immediately placed in the freezer at –20 °C.

Effects of simulated and real herbivory across a nutrient gradient

Because there were few pronounced effects of varying nitrogen availability in the first experiment, we changed the nutrient treatment in the second experiment to either the presence or absence of added nutrients. We added an additional treatment of herbivory with a generalist caterpillar in order to compare the effects of real and simulated herbivory. Plants were grown as in the previous experiment. Once the plants reached 12 weeks of age (approx. 20 leaves, 25 cm in height), we initiated our treatments, including herbivory (0 % removed, 50 % leaf area removed through simulated herbivory and 50 % leaf area removed through real herbivory) and nutrient availability (unfertilized and fertilized). A two-way factorial design was used with eight replicates per treatment combination for a total of 48 plants. The simulated herbivory treatment was carried out as before. Real herbivory was implemented by surrounding whole plants in sleeve cages and placing five to ten Hyphantria cunea (Actiidae) larvae collected from the source plant population on each plant. Larvae were allowed to feed for 3 d to achieve approx. 50 % defoliation on the entire plant. High-nutrient plants were fertilized with 125 mL of 1·87 g L−1 Peters 20–20–20 complete soluble fertilizer (240 ppm nitrogen) plus micronutrients (Grace-Sierra, Milpitas, CA, USA) and were watered between nutrient additions with distilled water. Low nutrient plants were watered with distilled water only. Growth responses (as in the first experiment) were taken every 2 weeks and harvest was conducted 10 weeks after initiation of treatments. To capture temporal differences in defence chemistry, leaf samples (approx. 500 mg mass) were randomly collected from each plant for chemical analysis 3 d after the caterpillar-defoliation treatments were completed and at the end of the experiment. Initial leaf samples from the caterpillar-defoliation treatments included leaves damaged by caterpillars; otherwise, whole leaf samples were taken from control plants or those receiving simulated herbivory. Leaf samples were immediately placed in the freezer at –20 °C until chemical analysis.

Chemical analyses

Resistance traits were evaluated in both experiments by comparing the leaves of damaged and undamaged plants for total protein content (which should increase with the accumulation of defence proteins), peroxidase activity (an oxidative enzyme associated with the synthesis or oxidative activation of defensive compounds) (Roitto et al., 2003; Cipollini et al., 2011) and total flavonoids (a class of phenolic compounds linked to multiple functions including anti-herbivore effects) (Cipollini et al., 2008b). Soluble proteins were extracted from fresh leaves by homogenizing leaves in ice-cold, 0·01 m sodium phosphate buffer (6·8 pH) containing 5 % polyvinylpolypyrrolidone. Extracts were centrifuged for 12 min at 9000 × g and the cleared supernatants transferred to fresh tubes. Total soluble protein was measured using bovine serum albumin as a standard and Bio-Rad protein dye reagent, as described in Bradford (1976). We used these estimates to calculate total soluble protein on a fresh mass basis (mg protein g−1 f. wt). Peroxidase activity was analysed in soluble protein extracts as in Cipollini et al. (2004) using guaiacol as the substrate. Peroxidase activities were expressed and statistically analysed as ΔAbs470nm min−1 mg extract protein−1. Soluble phenolic extracts were made by homogenizing fresh leaf material in 80 % methanol and shaking for 30 min on ice on an orbital shaker. Extracts were centrifuged as before and cleared supernatants were transferred to fresh tubes. Total flavonoid content was determined by mixing 0·2 mL of the methanol extracts with 0·01 mL of 1 % 2-amino-ethyl-diphenyl borate solution and analysed spectrophotometrically against a standard curve of apigenin at 404 nm (Hariri et al., 1991). Flavonoid concentration was expressed as milligram total flavonoids (apigenin equivalents) per gram fresh mass. Apigenin is a major flavonoid found in L. maackii leaves (Cipollini et al., 2008b). All assays were performed in duplicate.

Statistical analysis

For the first greenhouse experiment, independent and interactive effects of light availability, nitrogen availability and simulated herbivory on RGR in stem diameter, biomass and allocation data (total dry biomass, SLA and R:S), and chemistry data [total protein, peroxidase activity (POD) and total flavonoids] were examined using three-way factorial ANOVA. For the second greenhouse experiment, independent and interactive effects of fertilization and herbivory treatment on RGR and biomass data were analysed using a two-way factorial ANOVA. Chemistry data from the second experiment were examined using a three-way factorial ANOVA with the added factor of sampling time (either 3 d post-treatment or at the end of the experiment). Tukey post hoc testing was used to compare means. Total biomass, R:S, RGR, SLA, peroxidase activities and total flavonoids were log transformed and protein content was inverse transformed to meet the assumptions of normality. Pearson correlations were performed among all growth and chemical measures separately by each treatment combination in each greenhouse experiment. Root, shoot and total biomass of plants in both greenhouse experiments were significantly positively correlated, thus we only present the statistical results for total biomass. We also estimated the amount of biomass removed during the defoliation treatments based on the percentage of leaf area removed and the average leaf weight (taken from SLA calculations). Adding this biomass back to the shoot biomass of each plant had no effect on statistical patterns, so we show statistical results and means without the removed biomass included. All statistical analyses were performed using SAS (Version 9.2, SAS Institute, Cary, NC, USA).

RESULTS

Effects of simulated herbivory across light and nitrogen gradients

Both defoliation and light limitation reduced biomass accumulation, but there were no interactive effects (Table 1 and Fig 1A). With the exception of the high-light/low-nitrogen treatment, defoliation reduced total biomass between 12 % and 33 % (Fig. 1A). Defoliation reduced R:S, and there was an interaction between defoliation and light, and defoliation, light and nitrogen; the greatest change (3-fold increase) in R:S occurred in the high-light/high-nitrogen and low-light/low-nitrogen treatments (Table 1 and Fig. 1B). Defoliation and light limitation reduced RGR, and there was a significant interaction between defoliation, light and nitrogen (Table 1). The greatest difference in RGR was observed between undamaged plants in the high-light/high-nitrogen treatment and damaged plants in the low-light/low-nitrogen treatment (Fig. 1C). Overall, defoliation had no effect on SLA, which was influenced only by light limitation with higher values under low-light conditions (Table 1 and Fig. 1D).

Table 1.

Results of a three-way ANOVA evaluating the effects of light, nitrogen fertilization and simulated herbivory on growth and allocation patterns of juvenile Lonicera maackii plants

Response Effect d.f. F-value P-value
Total biomass Fertilizer 1 0·03 0·8603
Light 1 61·34 <0·0001
Fertilizer × light 1 3·51 0·0664
Defoliation 1 4·85 0·0318
Fertilizer × defoliation 1 0·72 0·3992
Light × defoliation 1 0·66 0·4194
Fertilizer × light × defoliation 1 1·95 0·1686
Root:Shoot Fertilizer 1 0·09 0·7712
Light 1 0·05 0·8263
Fertilizer × light 1 0·30 0·5887
Defoliation 1 15·11 0·0003
Fertilizer × defoliation 1 3·21 0·0785
Light × defoliation 1 5·29 0·0252
Fertilizer × light × defoliation 1 6·09 0·0167
RGR Fertilizer 1 0·08 0·7700
Light 1 6·58 0·0130
Fertilizer × light 1 0·01 0·9112
Defoliation 1 8·15 0·0060
Fertilizer × defoliation 1 0·58 0·4491
Light × defoliation 1 0·12 0·7325
Fertilizer × light × defoliation 1 5·24 0·0259
SLA Fertilizer 1 0·17 0·6796
Light 1 5·14 0·0275
Fertilizer × light 1 0·43 0·5135
Defoliation 1 0·85 0·3595
Fertilizer × defoliation 1 0·09 0·7617
Light × defoliation 1 0·42 0·5199
Fertilizer × light × defoliation 1 1·86 0·1781
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Fig. 1.

Fig. 1.

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(A) Total dry biomass, (B) root : shoot ratios, (C) relative growth rate of stem diameter at base (RGR DAB), and (D) specific leaf area of clipped and undamaged juvenile L. maackii plants grown in high (100 % ambient) or low (50 % ambient) light, and high (HN) or low (LN) nitrogen conditions. Means (± s.e.) are shown. Different letters indicate significant differences between high and low light treatments (P < 0·05).

Defoliation, light and nitrogen treatments had no significant main effects on total protein, POD or total flavonoid concentration in L. maackii plants, but the interaction of nitrogen and light significantly influenced protein and POD, and the interaction of defoliation and light influenced total flavonoid concentration (Table 2). Total proteins were highest in the high-light/low-nitrogen treatment and lowest in low-light/high-nitrogen treatment (Fig. 2A). Peroxidase activity was lowest in the low-light/low-nitrogen treatment, and did not vary among the other treatment combinations (Fig. 2B). Defoliation increased flavonoid concentrations under high-light conditions, but reduced them under low-light conditions (Fig. 2C). There were no correlations between total protein, POD or total flavonoid content with any measure of growth for damaged or undamaged plants (total biomass, R:S or RGR).

Table 2.

Results of a three-way ANOVA evaluating the effect of light, nitrogen fertilization and simulated herbivory on foliar chemistry of juvenile Lonicera maackii plants

Response Effect d.f. F-value P-value
Total protein Fertilizer 1 1·09 0·3021
Light 1 0·02 0·8839
Fertilizer × light 1 5·02 0·0294
Defoliation 1 0·47 0·4979
Fertilizer × defoliation 1 0·09 0·7691
Light × defoliation 1 2·39 0·1285
Fertilizer × light × defoliation 1 0·32 0·5751
POD Fertilizer 1 0·28 0·5965
Light 1 3·07 0·0854
Fertilizer × light 1 4·12 0·0475
Defoliation 1 1·47 0·2300
Fertilizer × defoliation 1 0·02 0·8920
Light × defoliation 1 3·14 0·0821
Fertilizer × light × defoliation 1 0·72 0·3986
Total flavonoid Fertilizer 1 0·74 0·3922
Light 1 0·14 0·7126
Fertilizer × light 1 2·82 0·0992
Defoliation 1 0·01 0·9431
Fertilizer × defoliation 1 1·03 0·3151
Light × defoliation 1 12·66 0·0008
Fertilizer × light × defoliation 1 2·82 0·0991
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Fig. 2.

Fig. 2.

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The effects of resource limitation (nitrogen and light) measured at the time of harvest (336 d old) on (A) total protein concentration, (B) peroxidase activity (POD) and (C) total flavonoid concentration of juvenile L. maackii plants subjected to clipping (simulated herbivory, at 0 or 50 %) at 84 d old. HN and LN indicate the high-nitrogen and low-nitrogen treatments, respectively. Because simulated herbivory had no significant effect on total protein concentrations and POD, means (±s.e.) are presented by nitrogen (HN and LN) and light (high and low) treatment. There was a significant interaction between light and simulated herbivory on total flavonoid concentrations. Therefore, means (±s.e.) of total flavonoid concentrations are presented by light (50 and 100 %) and simulated herbivory treatment.

Fig. 3.

Fig. 3.

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The effects of clipping and caterpillar defoliation on (A) total dry biomass, (B) root: shoot ratios, (C) relative growth rate of stem diameter at base (RGR DAB), and (D) specific leaf area of juvenile L. maackii plants grown in full- or no-fertilization treatments (HN and LN, respectively). Means (± s.e.) are shown. Different letters indicate significant differences between treatments (P < 0·05).

Effects of simulated and real herbivory across a nutrient gradient

Total biomass was influenced independently by defoliation treatment and R:S was influenced by fertilization treatment, but there were no interactions between defoliation and fertilization for either variable (Table 3). The mean total biomass of control plants was 1·6 times higher than plants receiving real herbivory. Plants receiving simulated herbivory did not differ from controls or those receiving real herbivory (Fig. 3A). R:S decreased with fertilization (Fig. 3B). There were no significant effects of the treatments on RGR or SLA (Table 3 and Fig. 3C, D).

Table 3.

Results of a two-way ANOVA evaluating the effect of defoliation treatment (control, simulated, real) and nitrogen availability on juvenile Lonicera maackii plants

Response Effect d.f. F-value P-value
Total biomass Defoliation 2 8·88 0·0007
Fertilizer 1 1·48 0·2310
Defoliation × fertilizer 2 1·71 0·1943
Root:Shoot Defoliation 2 1·5 0·2349
Fertilizer 1 27·55 <0·0001
Defoliation × fertilizer 2 2·85 0·0697
RGR Defoliation 2 1·16 0·3253
Fertilizer 1 1·15 0·2900
Defoliation × fertilizer 2 1·15 0·3278
SLA Defoliation 2 0·77 0·4616
Fertilizer 1 0·31 0·5816
Defoliation × fertilizer 2 0·1 0·9069
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Overall, POD (11·96 ± 2·23 initial, 5·91 ± 1·28 final ΔAbs470 min−1 mg protein−1) and total flavonoid concentration (6·42 ± 0·47 initial, 4·07 ± 0·25 final mg g−1 f. wt) decreased through time, but were not significantly affected by defoliation treatment or fertilization (Table 4). There was a significant interactive effect between time and defoliation on total protein (Table 4). Foliar protein increased through time, but the change was greater in both defoliation treatments than in the control (Fig. 4). There were no correlations between initial and final total protein, POD or total flavonoid content with any measure of growth for damaged or undamaged plants (total biomass, R:S or RGR).

Table 4.

Results of a three-way ANOVA evaluating the effect of sampling time, defoliation treatment (control, simulated, real) and nutrient availability on foliar chemistry of juvenile Lonicera maackii plants

Response Effect d.f. F-value P-value
Total protein Time 1 134·11 <0·0001
Defoliation 2 0·48 0·6232
Time × defoliation 2 3·32 0·0421
Fertilization 1 0·1 0·7566
Time × fertilization 1 0·02 0·8944
Defoliation × fertilization 2 0·18 0·8316
Time × defoliation × fertilization 2 0·97 0·3840
POD Time 1 4·79 0·0321
Defoliation 2 2·02 0·1407
Time × defoliation 2 0·6 0·5515
Fertilization 1 1·02 0·316
Time × fertilization 1 0·06 0·8083
Defoliation × fertilization 2 2·59 0·0827
Time × defoliation × fertilization 2 1·05 0·3560
Total flavonoid Time 1 12·92 0·0007
Defoliation 2 0·87 0·4243
Time × defoliation 2 0·53 0·5899
Fertilization 1 3·41 0·0698
Time × fertilization 1 1·53 0·2216
Defoliation × fertilization 2 0·42 0·6596
Time × defoliation × fertilization 2 1·44 0·2453
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Fig. 4.

Fig. 4.

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The effects of clipping and caterpillar defoliation on mean (± s.e.) total protein concentrations in L.maackii leaves in pooled treatments (fertilization was not significant). Damage was imposed when plants were 84 d old and protein measured 3 d after defoliation treatment (‘initial’) and at time of harvest (336 d old ‘final’).

DISCUSSION

Pre-reproductive, juvenile L. maackii plants exhibited a high degree of tolerance to the loss of up to 50 % of their leaves when measured 12 weeks after damage, but a similar amount of total damage inflicted by a generalist caterpillar had more negative effects. Additional bouts or years of major defoliation in this critical developmental period could eventually lead to chronic reductions in growth as productive tissues above-ground are removed and below-ground resources are exhausted. Time to reproductive maturity of L. maackii plants typically ranges from 4 to 7 years (D. Lieurance, pers. obs.), so we could not assess the effect of herbivory on reproductive output, which could have been affected more greatly than vegetative growth (Hanley and Fegan, 2007; Hanley, 2012). Lonicera maackii generally experiences much lower amounts of arthropod herbivory in the field than the level imposed here (3 % observed vs. 50 % imposed), but can occasionally receive higher amounts of herbivory. Although sensitive to the loss of 50 % of its leaf area to an herbivore in the greenhouse, it would appear that typical field levels of arthropod defoliation are unlikely to impact on the performance of plants in most environments where they grow. Additionally, resource limitation, specifically limiting light, slowed growth and negatively impacted Lonicera's ability to tolerate defoliation. These results are consistent with studies illustrating the shade intolerance of L. maackii (Luken and Mattimiro, 1991; Luken et al., 1995a, 1997b). For example, Luken and Mattimiro (1991) demonstrated that the negative effects of repeated clipping (100 % stem removal) of mature L. maackii shrubs over three consecutive years were greater in the shady forest understorey than in full light.

High tolerance to herbivory has been observed in many other woody invasive plants. Ashton and Lerdau (2008) showed that mature invasive vines, including the L. maackii relative, Lonicera japonica, were more tolerant than native congeners and con-familials to simulated herbivory; invasives showing an ability to quickly compensate for above-ground tissue loss and to maintain a stable R:S. Similarly, artificial defoliation did not affect growth rates of invasive Triadica sebifera seedlings (Rogers and Siemann, 2002), and neither timing nor type of herbivory (low intensity – chronic defoliation or high intensity – acute defoliation) nor resource limitation (nitrogen and light) affected growth of seedlings in greenhouse or field experiments (Rogers and Siemann, 2003). Even though we observed under-compensation in total biomass in response to herbivory in both experiments, the response of R:S and RGR of juvenile L. maackii tended to be consistent with these findings. Furthermore, R:S increased with caterpillar herbivory, clipping and nutrient limitation. Prioritizing the allocation of resources below-ground after defoliation or under nutrient limitation is a form of phenotypic plasticity that may increase nutrient acquisition and aid regrowth after defoliation (Karban and Baldwin, 1997; Orians et al., 2011).

In addition to resource availability (Cipollini and Bergelson, 2001; Barto et al., 2008), allocation to chemical defences is influenced by plant age and seasonality (Witzell et al., 2007; Elger et al., 2009), possible trade-offs with allocation to tolerance mechanisms (Núñez-Farfán et al., 2007), and experience of earlier herbivore attack (Karban, 2011). Our results did not support the prediction that defoliation would increase total protein, POD and total flavonoid content, other than an increase in flavonoids noted for young, defoliated plants in high-light conditions. Since we sampled for chemistry at the end of the first experiment long after treatments were imposed, we were likely not to detect much induction, but in the second experiment we sampled 3 d after damage occurred and still did not observe any change in plant biochemistry due to defoliation (but our measures of plant chemistry were relatively crude). There was also minimal support for our prediction that resource limitation would affect foliar chemistry, but we did find seasonal changes, with total flavonoids and POD declining, and total protein increasing through time. Cipollini et al. (2008b) showed how foliar concentrations of luteolin (a flavonoid-derived compound) and its glycoside derivative in mature L. maackii plants decreased during the growing season, paralleling changes observed here in young plants Additionally, with no correlations between total protein, peroxidase and total flavonoids with measures of growth in damaged or undamaged plants, there appears to be no trade-off between growth and the aspects of plant chemistry that we measured.

Strauss and Agrawal (1999) stress that evidence of tolerance to herbivory of plants may vary between studies employing simulated and real herbivory, as well as different types of herbivory (e.g. leaf or root feeding). Although plant responses to defoliation are likely to vary due to variation in damage type (chewing vs. clipping) and biochemical stimuli from salivary enzymes (Strauss and Agrawal, 1999; Musser et al., 2005; Wu and Baldwin, 2012), we found similar effects of caterpillar herbivory and artificial clipping on plant chemistry. However, more precise biochemical measures may have revealed differences. For example, separation of phenolic profiles may have revealed individual flavonoids that responded differently to herbivory type, while the total flavonoid concentration may not have varied. In contrast to clipping where we removed whole leaves, caterpillars caused partial damage on more leaves. This may explain why herbivory resulted in lower final biomass than clipping. A sudden one time removal may stimulate the plant to prioritize resources to re-growth rather than costly chemical defences, whereas real herbivory is more spread out, takes more time and other responses may be triggered to compensate or defend against herbivory (Cipollini and Sipe, 2001).

Conclusions

Because both tolerance and resistance mechanisms are thought to be costly to a plant, it has been suggested that some plants will exhibit a trade-off between the two in response to herbivore damage, especially when limited by resources (van der Meijden et al., 1988; Fineblum and Rausher, 1995). But many studies indicate plants may allocate resources to both strategies (Koricheva et al., 2004; Núñez-Farfán et al., 2007; Agrawal, 2010). Lonicera maackii appears to have the capacity to both resist and tolerate herbivory, thus tolerance and resistance are not mutually exclusive in this plant. But, considering the low amounts of herbivory incurred by L. maackii in the field, this trade-off is not an issue as currently observed levels of damage by arthropod herbivores does not appear to be significant enough to impact on the performance of the shrub in its invasive range.

ACKNOWLEDGEMENTS

We thank Wright State University Biological Sciences Department, the Ohio Board of Regents and the Ohio Plant Biotechnology Consortium for funding, Dan Romanek, Jon Ali, and Dan Davis for greenhouse and laboratory assistance. Suggestions from Mick Hanley and two anonymous reviewers greatly improved the manuscript.

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